The human gene encoding preproTRH (thyrotropin-releasing hormone) was assigned to chromosome 3, using human-Chinese hamster ovary somatic cell hybrids, analyzed by Southern hybridizations. Hybridization was carried out with a 32P-labeled human preproTRH cDNA labeled by the method of random priming. Hybridization of the cDNA probe to a human specific 4.8-kb DNA fragment of EcoRI-digested WBC DNA was used to localize the human preproTRH gene. No hybridization, by contrast, was seen with human preproTRH cDNA probe and hamster DNA after EcoRI treatment. Results from 29 somatic cell hybrids corroborated unequivocally that the human preproTRH gene can be assigned to human chromosome 3.